Chemokine receptors (CRs) have drawn much attention since their description as human immunodeficiency virus (HIV) co-receptors by several groups in 1996. Prior to that time, HIV tropism was defined as either macrophage (M)- or T cell (T)-tropic, which corresponded to non-syncytia- or syncytia-inducing viruses, respectively. Today, the classification of HIV tropism is defined by chemokine receptor usage of either CCR5, CXCR4, or both receptors. Chemokine receptors are a family of seven transmembrane spanning G protein-coupled receptors that are differentially expressed by a number of immune and non-immune cell populations. Certain CRs have been shown to be palmitoylated and targeted to cholesterol-and sphingolipid-rich membrane microdomains termed lipid rafts. Lipid rafts is a broad term for the collection of membrane microdomains enriched in cholesterol, sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and acylated signaling molecules. Lipid rafts are believed to be important signaling platforms enriched in many signaling proteins, including but not limited to src kinases, Ga subunit, H-Ras, LAT, and NOS. Signal transduction through the T and B cell receptors as well as the IgE receptor involves the recruitment of signaling assemblies to lipid rafts. CCR5 and CXCR4 have been shown to be present in lipid rafts, colocalizing at the leading edge of migrating cells. CCR5 has also recently been shown to be palmitoylated, which is one of the important modifications in lipid raft targeting of proteins. However, the role of cholesterol and these lipid rafts on T cell chemokine binding and signaling through CCR5 and CXCR4 remains unknown. We found that cholesterol extraction by beta-cyclodextrin (BCD) significantly reduced the binding and signaling of CXCR4 and MIP-1b using CXCR4- or CCR5-expressing T cells, respectively. Reloading treated cells with cholesterol but not 4-cholesten-3-one, an oxidized form of cholesterol, restored chemokine binding to BCD-treated cells. Antibodies specific for distinct CXCR4 or CCR5 epitopes lost their ability to bind to the cell surface after cholesterol extraction. Moreover, cells stained with fluorescently-labeled MIP-1b extensively co-localized with the GM1 lipid raft marker while using anti-CCR5 antibodies, the majority of CCR5 on these cells co-localized with CD59 and only partially with GM1 suggesting that active ligand binding facilitates receptor association with lipid rafts or that raft association promotes a higher affinity conformation of CCR5. Together, these data demonstrate that cholesterol and lipid rafts are important for the maintenance of the chemokine receptor conformation and are necessary for both the binding and function of this chemokine receptor. This cholesterol and lipid raft requirement for ligand binding may play a significant physiological role in controlling immune cell signaling and migration. Oxidized forms of cholesterol, known as oxysterols, are abundant in various food products and can be found naturally in membranes and mitochondria of a variety of cell types. Oxysterols have been reported to modulate a number of cellular functions and signals, including the inhibition of HMG Co-A reductase activity and sterol synthesis, increasing calcium influx, decreasing cholesterol efflux, the induction of cellular apoptosis, and the initiation of atherosclerosis. The incorporation of oxysterols, such as 22-hydroxycholesterol (22-OHC) and 25-OHC, into cell membranes has previously been shown to exert immunosuppressive effects on lymphocytes. Our results revealed that 22-OHC and 25-OHC, but not cholesterol, significantly reduced the binding of both SDF-1a and MIP-1? to T cells and PBMCs. These oxysterols also inhibited the intracellular calcium mobilization and chemotaxis in response to SDF-1a and MIP-1b treatment. The inhibitory effects of oxysterols were rapid and approached maximum levels within 1 hour of incubation. In contrast to the ligand binding studies, anti-CXCR4 and CCR5 antibody binding to T cells was not inhibited by oxysterol treatment. In addition, while the presence of oxysterols in cell membranes significantly inhibits chemokine receptor function, this effect does not involve alterations to receptor conformation, receptor internalization, or direct blocking of chemokine binding. We hypothesize that the increased presence of circulating and/or membrane-associated oxysterols in athlerosclerotic patients and in various chronic disease states may play a role in the diminished immune responses observed in elderly and frail subjects. Similarly, enzymatic cholesterol oxidation using cholesterol oxidase (CO) alone or in combination with sphingomyelinase (SMase) results in similar inhibitory effects as those observed with OHC treatment. CO is an enzyme that converts cholesterol (3b-hydroxy-5-cholestene) into 4-cholesten-3-one and is produced by various pathogens including Streptomyces, Pseudomonas, Mycobacterium, Brevibacterium, Nocardia, Rhodococcus and Corynebacterium. We have found that that CO treatment inhibits chemokine binding to CXCR4 and CCR5 on T cells, resulting in the significant inhibition of chemokine-mediated intracellular calcium mobilization and chemotaxis. Thus, we propose a novel mechanism for pathogen modulation of chemokine function in the immune cell response to infection and inflammation.